The present invention relates to a DNA sequence encoding a novel neuronal protein kinase (NPK) which phosphorylates tau proteins as well as other microtubule associated proteins (MAPs) in positions crucial for the binding to microtubules. The invention further relates to Serine or Theorine residues and epitopes comprising said residues phosphorylated by said NPK on said MAPs, to antibodies specifically binding to said protein kinase, pharmaceutical compositions comprising inhibitors to said protein kinase, in particular for the treatment of Alzheimer""s disease and cancer, to diagnostic kits and to in vitro diagnostic methods for the detection of Alzheimer""s disease and cancer.
Microtubule associated proteins (MAPs) regulate the extensive dynamics and rearrangement of the microtubule network which is thought to drive neurite outgrowth (reviewed recently by Hirokawa, 1994). Several lines of evidence suggest that the phosphodrylation state of MAPs, balanced by protein kinases and phosphatases in a hitherto unknown way, plays a pivotal role in the modulation of these events. Tau protein, a class of MAPs in mammalian brain (Cleveland et al., 1977), is phosphorylated on several sites in vivo (Butler and Shelanski 1986; Watanabe et al., 1993) and is a substrate for many protein kinases in vitro (reviewed by Lee, 1993; Goedert, 1993; Mandelkow and Mandelkow, 1993; Anderton, 1993). During neuronal degeneration in Alzheimer""s disease, tau protein aggregates into paired helical filaments (PHFs), the principal fibrous component of the characteristic neurofibrillary lesions (reviewed by Lee and Trojanowski, 1992). Tau isolated from these aggregates displays some biochemical alterations, of which hyperphosphorylation is the most striking (Grundke-Iqbal et al., 1986; Brion et al., 1991; Ksiezak-Reding et al., 1992; Goedert et al., 1992). Most of the reported aberrant phosphorylation sites are Ser/Thr-Pro sequences (Lee et al., 1991; Biernat et al., 1992; Lichtenberg-Kraag et al., 1992; Gustke et al., 1992; Watanabe et al., 1993), suggesting a dysregulation of proline-directed kinases (Drewes et al., 1992; Mandelkow et al., 1992; Hanger et al., 1992; Vulliet et al., 1992; Baumann et al., 1993; Paudel et al., 1993, Kobayashi et al., 1993) or the corresponding phosphatases (Drewes et al., 1993; Gong et al., 1994). Phosphorylation-dependent antibodies, which discriminate between xe2x80x98normalxe2x80x99 tau and the hyperphosphorylated, xe2x80x98pathologicalxe2x80x99 forms, were prepared by several laboratories (Kondo et al., 1988; Lee et al., 1991; Mercken et al., 1992; Greenberg et al., 1992). All of these antibodies were shown to be directed against epitopes of the Ser/Thr-Pro type (Lee et al., 1991; Biernat et al., 1992; Lichtenberg-Kraag et al., 1992; Lang et al., 1992; Watanabe et al., 1993).
The microtubule binding region of tau (FIG. 1) includes three or four pseudorepeats of 31 residues each depending on isoform type (Lee et al., 1989; Goedert et al., 1989; Himmler et al., 1989). This region probably forms the building block of the paired helical filaments (Kondo et al., 1988; Wischik et al., 1988; Ksiezak-Reding and Yen, 1991; Wille et al., 1992). It does not contain any of the 14-16 Ser/Thr-Pro motifs, which accumulate in the regions flanking the repeats. However, it contains a conserved Serine residue (Ser262) within the sequence KIGS in the first repeat, which was found to be one of the predominant sites phosphorylated by a tissue extract from brain (Gustke et al., 1992). This site is also found to be phosphorylated in Alzheimer PHF-tau, but not in xe2x80x98normalxe2x80x99 tau or fetal tau (Hasegawa et al., 1992). So far, it is the only pathological phosphorylation site found within the repeat domain of tau.
Recently, a site-directed mutagenesis approach was used to show that phosphorylation of tau at this site strongly decreases its microtubule binding capacity, whereas the phosphorylation on Ser/Thr-Pro motifs had only a minor effect (Biernat et al., 1993). This initiated a search for protein kinases in neuronal tissue with the ability to phosphorylate tau at Ser262. The technical problem underlying the present invention was to provide a protein kinase which is causative for the onset of Alzheimer""s disease by phosphorylating the crucial Serine 262 residue of human tau protein and a corresponding nucleotide sequence.
The solution to this technical problem is achieved by providing the embodiments characterised in the claims.
Thus, the present: invention relates to a DNA sequence encoding a neuronal protein kinase (NPK) or a functional fragment thereof that is capable of phosphorylating a sequence motive of the type KXGS in tau, MAP4, MAP2 and MAP2c characterised by the following features:
(a) it encodes the amino acid sequence depicted as MARK-1 in Table 6;
(b) it encodes the amino acid sequence depicted as MARK-2 in Table 6; or
(c) it hybridises to the DNA of (a) or (b).
The term xe2x80x9cDNA sequencexe2x80x9d comprises any DNA sequence such as genomic or cDNA, semisynthetic or synthetic DNA.
It was surprisingly found that none of the prior art kinases is mediating the phosphorylation of the four KXGS motifs in the repeat domain of tau to an extent that is sufficient to explain the biological and pathological effects associated with said phosphorylation. This is particularly true for Serine residue 262 which is indicative of the onset of Alzheimer""s disease. Instead, the present invention provides a DNA sequence encoding a novel protein kinase with the above identified features which is responsible for the phosphorylation of the amino acid residues crucial for the onset of Alzheimer""s disease. Said protein kinase is, also termed NPK, MARK-1 or MARK-2 throughout this application. The numbering of amino acid residues referred to in this application ensues with regard to the sequence of htau 40, the longest of the human tau isoforms (441 residues, Goedert et al., 1989).
In a preferred embodiment, the present invention further relates to a DNA sequence wherein the neuronal protein kinase (NPK) is characterised by the following features:
(a) it has an apparent molecular weight of 110 kD as determined by SDS-PAGE;
(b) it phosphorylates Serine residues 262, 293, 305, 324 and 356 of human tau protein; and
(c) it comprises the following amino acid sequences
KLDTFCGSPPYAAPELFQGK (SEQ ID NO: 1)
DRWMNVGHEEEELKPYAEP (SEQ ID NO: 2)
(K) SSRQNIPRCRNNI (SEQ ID NO: 3)
In a preferred embodiment of the DNA sequence of the present invention, the NPK is further characterised by the following features:
(d) it is deactivated by phosphatase PP-2A; and
(e) it phosphorylates the following Serine or Threonine residues of tau related microtubule-associated proteins (MAPs) MAP2, MAP2c and MAP4
MAP2/MAP2C: S37, S1536, S1676, S1707, S1792, S1796, S1799
MAP4: T829, T873, T874, T876, S899, S903, S928, S941, S1073
(f) it causes the dissociation of tau, MAP4, MAP2 and MAP2c from microtubules.
Another surprising finding that was made in accordance with the present invention is that the NPK by phosphorylating microtubule-associated proteins other than tau causes dissociation of these proteins from microtubules. This in turn results in the destabilisation of said microtubules, an increased dynamic instability thereof, and the ensuing effects on cell proliferation, cell differentiation, or cell degeneration. The NPK of the invention thus has the capacity to regulate the dynamics and rearrangements of microtubules in brain via the phosphorylation of tau or other MAPs. The finding referred to above has important implications for the role in the kinase of the invention in the generation of cancer.
This is because it is believed that cancer essentially is uncontrolled cell proliferation. Many anti-cancer drugs therefore interfere with cellular division and proliferation by poisoning the microtubules. On the other hand, xe2x80x9concogenesxe2x80x9d are often kinases, the cellular regulation of which is impaired. The deregulation of a kinase equal or homologous to the NPK of the invention could have serious effects on the stability of microtubules of various cell types. As microtubules play an important role in cell division, deregulation of said NPK can in turn lead to an uncontrolled cellular division and the transformation of normal cells to cancer cells. Alternatively, the deregulation of said NPK could provide postmitotic terminally differentiated cells such as neurons (which do not divide) with a stimulus to divide. This xe2x80x9cunnormalxe2x80x9d stimulus would lead the neurons directly into apoptosis (and thus, an Alzheimer""s like state) because due to their differentiation status they are unable to divide.
In a further preferred embodiment of the DNA sequence of the present invention, the NPK is obtainable from brain tissue by the following steps:
(a) homogenisation of brain extract and subsequent centrifugation thereof;
(b) chromatography of the supernatant obtained in step (a) on cellulosephosphate, wherein the NPK active fractions elute between 200 to 400 mM NaCl;
(c) ammonium sulfate precipitation of active fractions obtained in step (b) and dialysis of the precipitate;
(d) anion exchange chromatography of the dialysate obtained in step (c) on Q-Sepharose (Pharmacia) and elution of the NPK active fractions, wherein said NPK active fractions elute as a single peak at about 0.2 M NaCl, with subsequent dialysis of the active fractions;
(e) cation exchange chromatography on Mono S HR 10/10 (Pharmacia);
(f) chromatography on Mono Q HR 5/5, wherein the NPK active fractions elute at about 250 mM NaCl;
(g) gel filtration chromatography on Superdex G-200, wherein the NPK activity elutes with an apparent molecular weight of 100 kD; and
(h) affinity chromatography on ATP-cellulose, wherein the NPK active fractions elute with an apparent molecular weight of about 110 kD as determined by SDS-PAGE; wherein the NPK activity is measured by incubating a peptide comprising amino acid residues 255 to 267 of human adult tau in the presence of radioactively labelled ATP and determining the radioactivity incorporated into said peptide.
Further details as to how this NPK of the invention which in one embodiment has an apparent molecular weight of 110 kD (NPK-110) can be isolated are provided in Example 1. However, the person skilled in the art would know from the technical teaching given above how to supplement said details.
The NPK of the invention may be derived from any vertebrate brain. In a preferred embodiment, the NPK is derived from a mammalian brain.
The invention also relates to a RNA sequence complementary to the DNA sequence of the invention.
In a particularly preferred embodiment, said mammalian brain is human or porcine brain.
The invention further relates to a polypeptide encoded by the DNA sequence .or a functional fragment or derivative thereof. Said polypeptide, fragment or derivative may be posttranslationally or chemically modified. Throughout this specification, the term NPK or, alternatively, MARK (1 or 2) may also comprise such fragments or derivatives, even if this is not specifically indicated.
The present invention further relates to the following Serine or Threonine residues phosphorylated by NPK-110 of tau related microtubule-associated proteins (MAPs) MAP2, MAP2c and MAP4:
MAP2/MAP2c: S37, S1536, S1676, S1707, S1792, S1796, S1799
MAP4: T829, T873, T874, T876, S899, S903, S928, S941, S1073
and to epitopes comprising said phosphorylated Serine or Threonine residues.
The invention relates further to an antibody specifically binding to the NPK of the invention.
Said antibody may be a serum derived or a monoclonal, antibody. The production of both monoclonal and polyclonal antibodies to a desired epitope is well known in the art (see, for example, Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988). Furthermore, said antibody may be a natural or an antibody derived by genetic engineering, such as a chimeric antibody derived by techniques which are well understood in the art. Moreover, the term antibody as used herein also refers to a fragment of an antibody which has retained its capacity to bind the specific epitope, such as a Fab, F(ab)2 or an Fv fragment.
Additionally, the present invention relates to an antibody specifically binding to epitopes comprising the phosphorylated Serine or Threonine residues of MAP2, MAP2c and MAP4:
MAP2/MAP2c: S37, S1536, S1676, S1707, S1792, S1796, S1799
MAP4: T829, T873, T874, T876, S899, S903, S928, S941, S1073
Again, said antibody may be a polyclonal or a monoclonal antibody, or a fragment thereof retaining its binding specificity.
In a preferred embodiment, the antibody of the invention is a monoclonal antibody or a fragment or derivative thereof.
In a further preferred embodiment of the invention, said antibody is a polyclonal antibody or a fragment or a derivative thereof.
The invention furthermore relates to a pharmaceutical composition which contains a specific inhibitor of the NPK of the invention, optionally in combination with a pharmaceutically acceptable carrier and/or diluent.
The term xe2x80x9cspecific inhibitor of the NPK of the inventionxe2x80x9d refers to substances which specifically inhibit the enzymatic action of the protein kinase of the present invention. Inhibitors to enzymes such as protein kinases and their mode of action are well known in the art. For example, such an inhibitor may bind to the catalytic domain of the enzyme, thus rendering it incapable of converting its substrate.
Said pharmaceutical composition may be administered to a patient in need thereof by a route and in a dosage which is deemed appropriate by the physician familiar with the case. Pharmaceutically acceptable carriers and/or diluents are well known in the art, and may be formulated according to the route of administration or the special disease status of the patient.
In a preferred embodiment, the present invention relates to a pharmaceutical composition for the treatment of Alzheimer""s disease.
Again, said pharmaceutical composition may be administered to a patient in need thereof by a route and in a dosage which is deemed appropriate by the physician handling the case.
In a further preferred embodiment, the pharmaceutical composition of the present invention is used for the treatment of cancer.
As has been pointed out above, the deregulation of the NPK of the invention can lead a variety of cell types expressing microtubule associated proteins into a pathway that eventually results in the neoplastic transformation of said cells. Accordingly, a pharmaceutically effective amount of an NPK inhibitor will halt and/or reverse the transformation process. The amount of inhibitor to be administered will be determined by the physician handling the respective cases.
In a further preferred embodiment of the pharmaceutical composition of the invention, said inhibitor is the antibody of the invention, a phosphatase capable of dephosphorylating the NPK of the invention, preferably phosphatase PP-2A, an inhibitor of the activating kinase of said NPK, a tau derived peptide comprising the Ser262 residue or a MAP2, 2c or MAP4 derived peptide comprising at least one of the Serine or Threonine residues of MAP2, MAP2c or MAP4:
MAP2/MAP2c: S37, S1536, S1676, S1707, S1792, S1796, S1799
MAP4: T829, T873, T874, T876, S899, S903, S928, S941, S1073
The terms xe2x80x9ctau derived peptide comprising the Ser262 residue and a MAP2, 2c or MAP4 derived peptide comprising at least one of the Serine or Threonine residues of MAP2, MAP2c and MAP4:
MAP2/MAP2c: S37, S1536, S1676, S1707, S1792, S1796, S1799
MAP4: T829, T873, T874, T876, S899, S903, S928, S941, S1073xe2x80x9d
as used herein refers to a peptide which in its three dimensional structure reconstitutes the natural conformation of the tau protein or the MAP2, 2c or 4 proteins with regard to the epitope comprising Serine residue 262 (tau) or the other residues referred to above (MAP) MAP2, MAP2c and MAP4. These peptides will mimic the natural substrate (i.e. tau or tau related MAPs) of the NPK of the invention, but will not display any NPK associated biological effect. The synthesis of said peptides which solely may consist of the epitopes, or may comprise additional flanking amino acids, is well known in the art.
The present invention further relates to a diagnostic composition comprising:
(a) the NPK of the invention;
(b) the antibody or fragment or derivative of the invention; and/or
(c) apeptide comprising the phosphorylatable Serines or Threonines of tau, MAP2, MAP2c or MAP4 indicated above.
Said diagnostic composition may, for example, be used for the detection of Alzheimer""s disease or cancer or the onset thereof. The antibody of the invention may be used to detect abnormal, in particular higher concentrations or levels, of the NPK of the invention, a higher degree of activation of said NPK, which are indicative of said diseases. The NPK delivered with the composition could be used as an internal control. On the other hand, the above defined peptides may be used as substrates to detect an abnormal activity of the NPK of the invention. Again, the activity of the NPK comprised in the diagnostic composition may serve as an internal control.
The antibody specifically binding to the phosphorylated Serine residues enumerated above and comprised in MAP4, MAP2 or MAP2c may be used to detect an abnormal phosphorylation status or pattern of these microtubule associated proteins which is indicative of cancer.
Further applications of the diagnostic composition are as follows. Thus, in one embodiment, said diagnostic composition may comprise an antibody of the invention directed to one of the epitopes referred to above. For example, an Alzheimer""s or cancer correlated disease state of a sample may be detected by treating said sample with an antibody recognising one or more of said epitopes. The antibody-epitope (hapten) complex may be visualised using a second antibody directed to the antibody of the invention and being labelled according to methods known in the art (see, for example, Harlow and Lane, ibid.).
In still another embodiment of the present invention, said diagnostic composition may consist of an epitope referred to above and an antibody of the invention. Treatment of a sample with said antibody may give rise to conclusions with regard to the disease state of the corresponding patent, if the binding of said antibody to said sample is brought in relation to binding of said antibody to said epitope referred to above used as a reference sample.
In still another embodiment, the diagnostic composition may comprise an epitope referred to above, the NPK of the invention and an antibody of the invention. Kinase activity may be monitored with respect to phosphorylation of the sample as compared to the phosphorylation of the epitope of the invention. From the quantitated NPK activity the phosporylation state of the tau protein or the MAP2, 2c or 4 contained in said sample and therefore the disease state of the patient may be deduced. The kinase activity may, for example, be deduced by including a substrate analog in the same reaction, which is visually detectable upon enzymatic conversion. Such substrate analogs are widely used in the art. Alternatively, the amount of a phosphorylated tau protein or MAP2, 2c or 4 in the sample may be detected after treatment with the kinase of the invention by employing an antibody of the invention directed to the phosphorylated epitope and using the amount of antibody-epitope complex provided by the diagnostic composition as an internal standard, or by determining the amount of phosphate incorporated into tau protein or MAP2, 2c or 4 by the NPK, for example, by radioactive tracer methods which are well known in the art.
It should be kept in mind, however, that the person skilled in the art, being familiar with diagnostic principles, can easily combine the above mentioned compound in a different manner or supplement the composition with secondary or tertiary, labelled or unlabelled antibodies, or with enzymes and substrates. These embodiments are also covered by the present invention.
In still another embodiment, the invention relates to a method for the in vitro diagnosis and/or monitoring of Alzheimer""s disease comprising assaying a cerebrospinal fluid isolate of patient or carrying out a biopsy of nerve tissue (for example, olfactory epithilium) and testing said tissue for the presence of the NPK of the invention.
The invention further relates to a method for the in vitro diagnosis and/or monitoring of Alzheimer""s disease comprising assaying a cerebrospinal fluid isolate of a patient or carrying out a biopsy of nerve tissue and testing said tissue for the presence of unphysiological amounts of the NPK of the invention, or for unphysiological activity of said NPK.
An example of a nerve tissue suitable for said biopsy is the olfactory epithelium.
The method of the invention may, for example, be carried out by using the diagnostic composition of the invention, in particular the antibody directed to said NPK. Therefore, in a preferred embodiment of the invention, the NPK of the invention is detected by the antibody of the invention specifically binding to said NPK.
Additionally, the invention relates to a method for the in vitro diagnosis for cancer or the onset of cancer comprising assaying a suitable tissue or body fluid for the presence of phosphorylated Serine or Threonine residues of tau related microtubule associated proteins (MAPs) MAP2, MAP2c and MAP4 in the positions:
MAP2/MAP2c: S37, S1536, S1676, S1707, S1792, S1796, S1799
MAP4: T829, T873, T874, T876, S899, S903, S928, S941, S1073xe2x80x9c
or for the presence of unphysiological amounts of the NPK of the invention or an NPK specific phosphatase. It is understood that the phosphorylation status of the Serine or Threonine residues has to be an unphysiological one. Methods for determining such a phosphorylation status have been described in detail in PCT/EP 92 02 829, which is incorporated herein by reference.
The assay for said phosphorylated Serine or Threonine residues may, for example, be carried out using an antibody specifically detecting said phosphorylated residues or the epitopes comprising said residues.
The amount of the NPK in the sample may be measured by using antibodies specifically directed thereto or by measuring their activity using a suitable substrate, for example, a peptide comprising the above referenced Serine or Threonine in a non-phosphorylated state or any of MAP2, MAP2c and MAP4 in unphosphorylated state. Methods for measuring the phosphorylation status of proteins have been described in detail in PCT/EP; 92 02 829. The activity of the phosphatases, for example PP-2A, PPI or calcineurin may be tested by providing the substrate, NPK of the invention, for example, comprised in the diagnostic composition of the invention.
A suitable tissue or body fluid f or carrying out this in vitro method of the invention is cerebrospinal fluid, blood, biopsies of tissue (for example, liver or skin).
Still another object of the invention is to provide a method for the in vitro conversion of normal MAP2, MAP2c or MAP4 by the treatment with the NPK of the invention into proteins phosphorylated at positions:
MAP2/MAP2c: S37, S1536, S1676, S1707, S1792, S1796, S1799
MAP4: T829, T873, T874, T876, S899, S903, S928, S941, S1073xe2x80x9d
said phosphorylation status being indicative of cancer or the onset of cancer. The conditions allowing the phosphorylation of""said MAPs can be determined by following the general teachings provided by the present application. The phosphorylated MAPs can then be recognised by specific antibodies. The results of said in vitro method will allow further insights into the generation of cancer.
Moreover, inhibitors may be tested which prevent the conversion of normal to MAP protein phosphorylated in the positions indicated above. These xe2x80x9cinhibitorsxe2x80x9d may be specific for the epitope to be phosphorylated by, for example, blocking the epitope, or may be directed to various domains on the protein kinase of the invention, NPK, as long as they prevent or disturb its biological activity. Another type of inhibition is the antagonistic action of phosphatases on said MAPs or said NPK, or the inhibition of the activating kinase of said NPK. Furthermore, the MAP generated by the method of the present invention may be employed in binding studies to microtubule structures in vitro and in vivo, thus contributing to the elucidation of the molecular basis underlying cancer.
The present invention relates, moreover, to the use of the phosphorylated Serine or Threonine residue(s) of the MAP of the invention or the epitope comprising said residue(s) for the generation of specific antibodies indicative of cancer or the onset of cancer.
The methods for obtaining said antibodies are well known in the art; thus, the generation of polyclonal or monoclonal antibodies may be conducted using standard methods (see, for example, Harlow and Lane, ibid.). If an oligo- or polypeptide is used for the generation of antibodies, it is desirable to couple the peptide comprising the epitope to a suitable carrier molecule capable of inducing or enhancing the immune response to said epitope, such as bovine serum albumin or keyhole limpet hemocyanin. The methods of coupling hapten (comprising or being identical to the epitope) and carrier are also well known in the art (Harlow and Lane, ibid.). It is also to be understood that any animal suitable to generate the desired antibodies may be used therefor.